Cercospora zeina, is a threat to maize production globally. Understanding the molecular basis for quantitative resistance to GLS is therefore important for food security. We developed a de novo assembly pipeline to identify candidate maize resistance genes. Near-isogenic maize lines with and without a QTL for GLS resistance on chromosome 10 from inbred CML444 were produced in the inbred B73 background. The B73-QTL line showed a 20% reduction in GLS disease symptoms compared to B73 in the field (p = 0.01). B73-QTL leaf samples from this field experiment conducted under GLS disease pressure were RNA sequenced. The reads that did not map to the B73 or C. zeina genomes were expected to contain novel defense genes and were de novo assembled. A total of 141 protein-coding sequences with B73-like or plant annotations were identified from the B73-QTL plants exposed to C. zeina. To determine whether candidate gene expression was induced by C. zeina, the RNAseq reads from C. zeina-challenged and control leaves were mapped to a master assembly of all of the B73-QTL reads, and differential gene expression analysis was conducted. Combining results from both bioinformatics approaches led to the identification of a likely candidate gene, which was a novel allele of a lectin receptor-like kinase named L-RLK-CML that (i) was induced by C. zeina, (ii) was positioned in the QTL region, and (iii) had functional domains for pathogen perception and defense signal transduction. The 817AA L-RLK-CML protein had 53 amino acid differences from its 818AA counterpart in B73. A second “B73-like” allele of L-RLK was expressed at a low level in B73-QTL. Gene copy-specific RT-qPCR confirmed that the l-rlk-cml transcript was the major product induced four-fold by C. zeina. Several other expressed defense-related candidates were identified, including a wall-associated kinase, two glutathione s-transferases, a chitinase, a glucan beta-glucosidase, a plasmodesmata callose-binding protein, several other receptor-like kinases, and components of calcium signaling, vesicular trafficking, and ethylene biosynthesis. This work presents a bioinformatics protocol for gene discovery from de novo assembled transcriptomes and identifies candidate quantitative resistance genes.Gray leaf spot (GLS) disease in maize, caused by the fungus  Maize is one of the three main cereal crops that are globally used as a food source and is especially important in regions such as Africa, where it is a staple food . It contCercospora zeina  have been deployed to identify SNPs associated with QDR, such as a glutathione-and SCLB . Combiniand SCLB .However, the ultimate marker is within the causal gene . The mosC. zeina infection, and contrasting levels of kauralexins were found in resistant and susceptible RILs from the mapping population  analysis, and co-expression analysis on a population segregating for disease resistance . This wapulation .The genome sequence of maize line B73  and othede novo into transcripts. These studies identified 157, 1321, and 2355 novel transcripts from 6, 21, and 368 diverse inbred maize lines, respectively (Further evidence for diversity of gene content in maize germplasm has come from RNAseq data. Three studies conducted RNAseq on diverse maize lines and identified novel (non-B73) transcripts by mapping the reads to the B73 genome, collecting the unmapped reads, and assembling these ectively . These fC. zeina is also available, so the reads can be simultaneously mapped to this genome, allowing removal of the fungal reads. The reads that are left are potentially derived from the QTL, and these can be de novo assembled and annotated to identify candidate genes.This provides the rationale for this study in which a transcriptomics approach is used to contrast gene expression between near-isogenic lines in the B73 background that differ only in the QTL locus from a diverse maize source. The hypothesis is that transcripts derived from B73 will be common to both NILs and that these can be discarded after mapping reads to the high-quality single-molecule sequenced B73 genome. In addition, the genome sequence of the pathogen C. zeina, and unmapped reads were de novo assembled. We also conducted differential gene expression analysis on control and C. zeina-challenged leaf material. Our approach led to the identification of an allele of a lectin receptor-like kinase from CML444 positioned in the QTL region and induced by the pathogen as the most likely candidate gene, as well as a range of other defense-related transcripts that could contribute to the QDR to GLS disease.In this study, we applied this approach to a GLS QTL on chromosome 10 that we had previously identified in maize inbred line CML444 . Near-isThe maize material used in this study was comprised of near-isogenic lines in the inbred B73 background. A previous QTL mapping study of a RIL population derived from the parents CML444 and SC Malawi identified a GLS disease severity QTL on chromosome 10 . In thatRRID:SCR_006472); however, the current study used data from the B73-QTL plants.The B73 and B73-QTL NILs were planted at Greytown, KwaZulu-Natal, South Africa in a randomized block design with three replicate blocks of ten plants per row each. This site is a hotspot for GLS disease. GLS disease and RNAseq data from the B73 plants in this field trial have been reported by The GLS disease severity of the B73 and B73-QTL maize plants was assessed using a 1–9 scale as reported in C. zeina gDNA in the same leaf samples was quantified by quantitative PCR (qPCR) as a proxy for the fungal biomass, using the methods described in C. zeina-challenged” samples. It was expected that the lower leaves would be subjected to greater C. zeina challenge than the upper leaves, on account of epidemiological knowledge of the disease that inoculum is mainly derived from maize stubble from the previous season  were collected, with upper and lower leaf samples taken from the same plant. Two leaves per treatment were sampled to make up a biological replicate; for example, B73-QTL control “replicate one” was made up of a pool of two upper leaf pieces from B73-QTL plant 1, and two lower leaves of the same plant were sampled and pooled for the B73-QTL C. zeina-challenged “replicate one.” The statistical significance of the differences between B73 and B73-QTL disease severity data was determined by Student’s t-tests at a 95% confidence interval in R .GLS disease severity was also assessed using digital image analysis to calculate disease lesion area, and the amount of s season . HoweverC. zeina gDNA by qPCR and RNA extraction methods have been described for the B73 samples in RRID:SCR_011114) on an Illumina HiSeqTM 2000 instrument with 200 bp insert libraries and a 100 bp paired-end module . This resulted in six datasets of RNA-seq reads made up of three replicates of the B73-QTL control and three replicates of the B73-QTL C. zeina-challenged leaves. The B73 RNAseq dataset has been reported in The same B73-QTL leaf samples from the field trial carried out at Greytown, South Africa described above and used for GLS disease severity assessment were processed for RNA sequencing. The sub-sampling to quantify de novo assembly of the unmapped reads into transcripts; (iii) annotation of the de novo assembled transcripts; and (iv) differential expression analysis. The last step involved constructing a “master assembly” of all plant and fungal reads from a pool of all six B73-QTL samples to provide a reference to which reads in each replicate could be mapped, followed by differential gene expression analysis (The bioinformatics pipeline involved the following main steps: (i) identifying RNAseq reads that did not map to the maize or fungal genomes; (ii) analysis .RRID:SCR_014583) (RRID:SCR_011848)   to ident_011848) . Lower-qC. zeina CMW25467 genome sequences. Unmapped reads were identified from the B73-QTL control separately from the B73-QTL C. zeina-challenged samples by pooling the RNAseq reads from the three replicates of each treatment prior to mapping to the “maize/Cz reference sequence.” TopHat2  (ftp://ftp.ensemblgenomes.org/pub/plants/release-34/fasta/zea_mays/ (C. zeina draft genome (GenBank accession MVDW01000014.1). TopHat 2 was chosen for the mapping since it was designed to map reads to the main transcripts as well as splice variants (de novo assembly.The filtered B73-QTL RNA seq reads were mapped simultaneously to a combined “maize/Cz reference sequence” of both maize B73 and _013035)  with defea_mays/  and the variants , thus ende novo assembled using Trinity (RRID:SCR_013048) (de novo assembly were optimized using only the reads that mapped to the B73 genome (de novo assembly parameters used for assembly of the unmapped reads from the B73-QTL samples were the default parameters with k-mer size = 31.The unmapped reads produced by TopHat2 were converted to FASTA format using the FASTX-toolkit  and were_013048) . First, 3 genome . The optC. zeina-challenged) were determined using TransDecoder (RRID:SCR_006472) nr protein database using BLASTp with an e-value cut-off of 0.01.The open reading frames in each of the two Trinity FASTA files  and lower  leaves of the same plants, there are three potential factors that could result in differential expression of a gene: (i) induction by C. zeina, (ii) developmental differences between upper and lower leaves, or (iii) systemic responses in the upper leaves from the C. zeina-challenged lower leaves.The main aim was to determine if any of the novel transcripts from the unmapped RNAseq reads were differentially expressed between control B73-QTL and de novo assemblies could not be compared directly, since not all transcripts were assembled fully in both de novo assemblies. Therefore, a de novo “master assembly” was compiled from all reads from the experiment to serve as a reference for mapping and counting the reads from each biological replicate. Trinity (with the same parameters as described above) was used to compile the “master assembly” from a pool of the quality-filtered RNAseq reads from the three B73-QTL control replicates and the three B73-QTL C. zeina-challenged samples.The two separate RRID:SCR_017036) software   . The trig method .C. zeina (RRID:SCR_011919)  were determined by comparing them to the B73 v.4  and C. zC. zeina  referenc_011919) . Transcr1 (Transcripts classified as either B73 or with similarity to plant genes were uploaded into the Monocots PLAZA 4.0 workbench (with B73 v4 annotations)1  for annoC. zeina. A previous study . This data was compared to the expression profiles of B73 genes in B73-QTL plants that were found to be differentially expressed in response to C. zeina. GO enrichment analysis of the B73-QTL differentially expressed genes was carried out in PLAZA 4.0 using the hypergeometric distribution, and the Bonferroni method was applied to correct for multiple testing. RNAseq data for the six replicates of B73-QTL have been deposited in the GEO database (GSE137198).Another aim of the differential gene expression analysis was to compare global gene expression of maize genes between B73 and B73-QTL plants challenged with us study  had examde novo assembly of unmapped reads, and (ii) differential expression analysis after mapping to a master assembly, were combined at follows.The results of the two different analyses to identify QTL candidate genes in the B73-QTL plants, namely (i) de novo from the unmapped reads were matched to the transcripts in the master assembly by BLASTn of the B73-QTL master assembly transcripts against the B73-QTL control and B73-QTL C. zeina-challenged unmapped and de novo assembled transcripts. Matches with over 90% identity across over 90% of the query length were defined as the same transcript in the two datasets. Finally, a single combined table was compiled that listed the B73-QTL transcripts with B73-like or plant annotations in the following categories: (i) master assembly transcripts that were differentially expressed, and (ii) transcripts from the de novo assemblies of unmapped reads. All the bioinformatics scripts have been deposited at https://zenodo.org/record/3384940. This repository also includes the FASTA files of all the B73-QTL de novo assembled transcripts and predicted proteins listed in the combined table.Transcripts assembled de novo assembly of unmapped reads and the master assembly from B73-QTL that matched the lectin receptor-like kinase gene encoded by B73 Zm00001d026382 were combined. Sequence and annotation information of Zm00001d026382 was obtained from B73 RefGen_v4 at MaizeGDB2 (RRID:SCR_006600) (de novo assembled. These transcripts were then combined with those from the unmapped reads and master assembly of B73-QTL to obtain the full-length transcript of the gene from B73-QTL.The transcripts from _006600) . Since rde novo-assembled transcript from B73-QTL. CLC Main Workbench 8.0.1  and Primer Designer  were used to design the assay. PCR was conducted with the primers Lectin-F and Lectin-R (BamHI and HphI). PCR amplification conditions were: 3 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, and a final extension step of 5 min at 72°C. Sanger sequencing of the PCR products, restriction enzyme digestions, and visualization of the products by agarose gel electrophoresis were carried out using standard protocols.The PCR-RFLP assay to distinguish between the alleles of the lectin receptor-like kinase gene in B73 and CML444 was designed from an alignment of the B73 Zm00001d026382 gDNA sequence and the corresponding Lectin-R  to amplide novo assembled transcript of the lectin receptor-like kinase (variant of B73 Zm00001d026382) using BLASTn to identify putative syntelogs. Some of the genomes were not annotated, so regions with positive hits were manually annotated by identification of open reading frames using CLC Main Workbench. A synteny diagram was constructed with iADHoRe (within PLAZA) between genomic regions containing the putative syntelog of the B73-QTL lectin receptor-like kinase and several upstream and downstream gene models.Genome sequences of other maize lines and the wild relative teosinte available on MaizeGDB and sorghum available on NCBI (GCF_000003195.3) were queried with the B73-QTL C. zeina isolate CMW25467 was conducted using the protocol described in 2 on leaf four from the base of each maize plant was marked and inoculated with 1 × 106C. zeina conidia per milliliter using a soft paintbrush on replicate plants. Mock-inoculated plants were treated with water and kept in a separate section of the glasshouse. At 43 days post-inoculation (dpi), the maize plants were at the R1 stage of development, and mature GLS lesions were observed on the susceptible B73 plants. At this time point, all marked leaf samples were collected and flash-frozen in liquid nitrogen and stored at −80 prior to RNA extraction. Four biological replicate plants of each treatment were sampled, i.e., C. zeina-inoculated B73, C. zeina-inoculated B73-QTL, and mock-inoculated plants of both genotypes.The maize glasshouse inoculation experiment with Maize RNA was extracted from the B73 and B73-QTL leaf samples from the glasshouse trial treatments using QIAzol lysis reagent  according to the manufacturer’s instructions. A TURBO DNA-free kit  was used to remove any contaminating genomic DNA. cDNA synthesis was carried out with a Maxima H Minus First Strand cDNA Synthesis Kit  following the manufacturer’s instructions. Each reverse transcriptase reaction contained 2 μg RNA. RT-PCR was conducted with a primer pair in exon 4 and exon 7 regions that were identical between the CML444 and B73 (Zm00001d026382) full-length L-RLK transcripts  . An intrRT-qPCR was performed according to the MIQE guidelines. Primer Designer 4  was used to design primers. Three sets of primers were designed to distinguish expression between the CML and B73 copy of the L-RLK in B73-QTL plants and to profile the fully spliced variants. First, primers in the conserved exon 7 were used to measure the expression of both gene copies together (L-RLK e7 F1 and L-RLK e7 R1) . Second,TM Universal SYBR® Green Supermix , 10 μM of each primer, 100 ng of template cDNA, and sterile distilled water. The PCR cycling conditions were as follows: 95°C for 30 s, followed by 35 cycles of 95°C for 15 s and 60°C for 30 s. RT-qPCR data were analyzed using qbase + software, version 3.0 3. Statistical significance was evaluated by one-way ANOVA with a Tukey-Kramer post hoc test in qbase + software.Primer specificity was tested using conventional PCR, assessing the melting curves, and sequencing the RT-qPCR products. RPOL (DNA-directed RNA polymerase – Zm00001d012857) was used as a reference gene, using primers developed for normalization by p = 0.01, C. zeina inoculum on debris in the soil, and the pathogen progresses via rain splash upward through several infection cycles as the plant matures (p < 0.05)   . C. zein < 0.05) .C. zeina-challenged leaf samples produced 24–28 million reads per biological replicate   . A total3 genome .C. zeina-challenged samples, respectively, with 16 present in both  (data not shown). The differential expression analysis identified which transcripts were induced or repressed in the B73-QTL C. zeina-challenged leaves compared with the control. All transcripts in the master assembly were annotated in three categories – B73 genes, B73-like genes, “plant” genes, fungal genes, and “other” genes. A total of 5236 B73 and B73-like maize genes, 47 plant genes, 209 fungal genes, and 100 other genes were found to be significantly differentially expressed (FDR < 0.05).The strategy followed was to construct a single transcript “master assembly” from all B73-QTL samples and then map the reads from each replicate to the master assembly and conduct differential expression analysis. After C. zeina-challenged leaves showed overrepresentation of the GO terms related to responses to biotic stress  from B73 (C. zeina (Three different transcripts from the B73-QTL unmapped from B73 . FurtherC. zeina . The B73de novo assemblies (Acc# MT108451), and this was predicted to encode an 817 AA protein  with 93.5% identity to the 818 AA protein from B73  (A full length 2811 nt transcript was constructed from the B73-QTL 28_P002) . L-RLK-C28_P002) .l-rlk-cml transcript in B73-QTL was verified by inspection of the RNAseq reads that mapped to the Zm00001d026382 genomic DNA sequence. High coverage was observed across all seven predicted exons encoding the 818AA protein B73 v5 L-RLK P2   . The expeld data . In contperiment     (Supplem < 0.05) . No expr < 0.05) . The B73genotype . Sanger Synteny analysis was carried out, since one of the explanations for the two alleles of the lectin receptor kinase in CML444 (and B73-QTL) is adjacent gene duplication. This region of chromosome 10 shows synteny between five maize inbred lines, teosinte, and sorghum, with a BLH transcription factor and nuclear gene on one side and a protein kinase on the other . Maize CArabidopsis thaliana to the L-RLK-CML, and a phylogenetic tree was constructed using the maximum likelihood method  de novo assembly of unmapped reads identified a candidate lectin receptor-like kinase named L-RLK-CML derived from the QTL10 region; (iii) expression of the lectin receptor-like kinase was correlated with C. zeina pathogen load and induced in B73-QTL plants but not in B73 lacking the QTL; (iv) several other B73-like or novel maize transcripts with a potential role in QDR conferred by QTL10 were identified.The main findings from our implementation of this pipeline to B73 plants introgressed with QTL10 for GLS disease resistance from inbred CML444 were: (i) B73-QTL was more resistant to GLS caused by the pathogen de novo assembled transcript (l-rlk-cml), the candidate gene identified in this study. It encodes L-RLK-CML, an 817 AA lectin receptor-like kinase made up of a lectin ectodomain, a transmembrane domain, and an intracellular kinase domain with an active site signature (QIH29483). Gene-specific RT-qPCR showed that this copy was induced four-fold by C. zeina challenge. In contrast, the B73-like copy in B73-QTL was not induced by the pathogen. Similarly, in B73 plants, the single copy full-length transcript (Zm00001e041728_T002) encoding the full-length 818 AA protein (B73 v5 L-RLK P2) was expressed at a low level and not induced by C. zeina infection [an observation made from both field (RNAseq) and glasshouse (RT-qPCR) experiments].Further investigation revealed that two copies of the lectin receptor-like kinase gene are present in CML444 and B73-QTL in contrast to there being a single gene in B73. The main copy corresponded to the Genome annotations of this lectin receptor-like kinase gene in B73 had computationally predicted splice variants. Our RT-PCR analysis showed that the major transcripts produced in both B73-QTL and B73 with or without fungal challenge corresponded to the fully spliced transcript that coded for the full-length proteins with all functional domains (817AA L-RLK-CML in B73-QTL and 818AA B73 v5 L-RLK P2 in B73). We found no evidence for the alternative splice variant (v4 annotation) encoding a 698AA protein B73 v4 L-RLK P1 (AQK46433) lacking the serine/threonine kinase active site signature. Our data are consistent with the fact that this splice variant was removed from v5 of the genome annotation. The only other splice variant in the v5 annotation had retention of intron 5, resulting in a stop codon immediately after exon 5 and a protein truncated by 152 AA at the C-terminus. Our RT-PCR analysis showed minor products corresponding to incomplete splicing of either one or two of the three introns between exon 4 and exon 7, which would include this splice variant. The biological significance of these products is questionable, as they encode non-functional proteins and are thus unlikely to play a positive role in disease resistance conferred by QTL10. A survey of splicing in B73 maize revealed that intron retention accounted for 56% of alternative splicing events . Intron The candidate gene identified in this study encoding L-RLK-CML had 53 non-synonymous and 16 synonymous mutations compared with the B73 counterpart Zm00001e041728_T002 encoding B73 v5 L-RLK P2, which was annotated as a G-type lectin S-receptor-like serine/threonine-protein kinase. Domain analysis of L-RLK-CML showed that it contained all the functional domains of a G-type lectin receptor-like kinase . G-type receptor-like kinases (previously categorized as Bulb- or B-type due to their frequent occurrence in bulb species) are one of three classes of lectin receptor-like kinases . The besFusarium oxysporum f.sp. matthioli , but SD1-7 falls in the same clade and SD1-2 is in an adjacent clade to SD1-8 [according to  by cold .We noted that B73-QTL plants had two copies of the L-RLK gene, the candidate allele encoding L-RLK CML and a B73-like gene. This is in contrast to B73, which only has one copy. One explanation is that B73-QTL is heterozygous for this locus; however, this is highly unlikely for several reasons. First, the parental line CML444, which is inbred , also haC. zeina infection.The observation that the CML444 allele is most similar to teosinte and sorghum orthologs indicates that this allele might be older than the B73 allele. After duplication, it may have undergone subfunctionalization or neofunctionalization , and oneC. zeina, it is possible that the QDR may depend on another of the candidates identified in this study. Even though their closest B73 match is not positioned within the QTL region, these candidates may actually be within the QTL in CML444 due to differences in genomic organization compared to B73 . Ethylene is an important defense hormone, especially against necrotrophic pathogens, and therefore it is notable that a key enzyme in ethylene biosynthesis 1-aminocyclopropane-1-carboxylate oxidase was included  was implicated in immune responses and suppression of ABA signaling . The linincluded . PR protincluded .The limitations of our study, which fall outside of the scope of this work, as it focuses on bioinformatics applications in molecular plant pathology, are the provision of further biological experimental evidence of the candidate genes, especially the lectin receptor-like kinase. The methods to be followed to obtain this evidence would be (i) further fine-mapping of the QTL region to reduce the potential number of candidate genes, and (ii) knockout of candidate genes. Unfortunately, the maize mutant library resources, such as UniformMu in the W22 background, do not currently have a mutant in the gene model Zm00001d026382 . AnotherZmWAK, wheat Yr36, genes at the soybean rhg1 locus, and rice RKS1  position within the QTL region, (ii) plant immunity-related annotation, and (iii) induction by the pathogen. However, it should be borne in mind that the latter two criteria should be applied with care. For example, a QTL causal gene could have a novel function . Howeverice RKS1 .Finally, our study has provided a bioinformatics pipeline for identification of candidate genes underlying a QTL and has revealed the lectin receptor-like kinase as the strongest candidate, with additional alternative candidates. Lectin receptor-like kinases are currently a hot topic in plant immunity . MoreoveThe datasets generated for this study can be found in the NCBI GEO (Acc. GSE137198), NCBI (Acc. MT108451 and QIH29483) Zenodo, DB conceived the study. DB, YV, and RP participated in the design and management. YV and LS provided the resources. TW and LS performed the bioinformatics analyses. TW, VS, and KS conducted the experiments. TW and DB interpreted the data and wrote the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pre-CSD, mice susceptible to stress displayed increased fragmentation of Non-Rapid Eye Movement (NREM) sleep, due to increased switching between NREM and wake and shorter average duration of NREM bouts, relative to mice resilient to stress. Logistic regression analysis showed that the pre-CSD sleep features from both phenotypes were separable enough to allow prediction of susceptibility to stress with >80% accuracy. Post-CSD, susceptible mice maintained high NREM fragmentation while resilient mice exhibited high NREM fragmentation, only in the dark. Our findings emphasize the putative role of fragmented NREM sleep in signaling vulnerability to stress.There is a tight association between mood and sleep as disrupted sleep is a core feature of many mood disorders. The paucity in available animal models for investigating the role of sleep in the etiopathogenesis of depression-like behaviors led us to investigate whether prior sleep disturbances can predict susceptibility to future stress. Hence, we assessed sleep before and after chronic social defeat (CSD) stress. The social behavior of the mice  Global sleep problems are on the rise and negatively impact the quality of life and health, leading to chronic diseases including mood disorders. Common life stresses lead to sleep impairments such as reduced sleep time, compromised sleep consolidation and increased REM intensity . MoreoveSleep is a complex biological function that induces neural plastic changes in the brain . Accordiprior to CSD. Thus, we examined sleep in adult C57BL/6J male mice pre- and post-exposure to a 15-d CSD paradigm. Moreover, homeostatic sleep response following 4-h sleep deprivation (SD) was also assessed pre- and post-exposure to chronic social stress. Our findings demonstrate that mice that become susceptible to CSD stress exhibit pre-existing abnormal sleep/wake characteristics prior to stress exposure. Moreover, subsequent exposure to stress further impairs sleep and the homeostatic response.Repeated exposure to social stress, during the chronic social defeat (CSD) stress paradigm, is a commonly used rodent model of stress as it leads to continuous activation of the hypothalamic-pituitary-adrenal (HPA) axis and induction of depressive-like phenotypes such as social avoidance, anhedonia and anxiety . ChronicAll experiments performed were approved by the NYUAD Animal Care and Use Committee, and all experimental protocols were conducted according to the National Institute of Health Guide for Care and Use of Laboratory Animals (IACUC Protocol: 150005A2).ad libitum access to food and water in temperature (21 ± 2°C)- and humidity (50 ± 10%)-controlled facilities with 12-h light-dark (L/D) cycles . Zeitgeber time is a unit of time based on 12:12 light: dark cycle. All behavioral experiments were conducted during the light cycle (ZT 5 to ZT 10).CD1 retired breeders , and C57BL/6J male mice  were used in this study. All mice were maintained in the home cages, with Chronic social defeat stress paradigm was performed according to previously published protocols . The CD1On recovery day 16, social-avoidance behavior toward a novel non-aggressive but active CD1 mouse was measured in a two-trial social-interaction test. In the first 2.5 min trial, the experimental mouse was allowed to freely explore a square-shaped arena (44 cm × 44 cm) containing a perforated plexiglass cage (10 cm × 6 cm) along on one side of the arena (“No target” condition). In the second 2.5 min trial, the experimental mouse was reintroduced back into the arena with an unfamiliar CD1 non-aggressive mouse placed in the plexiglass cage (“Target” condition). Between trials, the behavioral apparatus was cleaned with MB-10 solution  to avoid persistence of olfactory cues. TopScan video tracking system (CleverSys. Inc.) was used to automatically monitor and record the amount of time the experimental mouse spent in the “interaction zone” (14 cm × 26 cm) interacting with the unfamiliar CD1 mouse, “corner zone” (10 cm × 10 cm) and “total travel” within the arena for the duration in both trials. Interaction zone time, corner zone time, total distance traveled were collected and analyzed. The classification of susceptible and resilient mice was based on the SI ratio, which was calculated as [100 × /] as described previously . All mic–1) and xylazine (10 mg kg–1) intraperitoneally. The mice were fixated in a stereotactic frame (Kopf instruments) at a sufficient level of anesthesia. The head was shaved, and the scalp was opened medially and the periosteum was removed. We used a dental precision driller (Stoelting) to drill four holes into the skull. The EEG electrodes were placed in the left and right part of the parietal lobe  and the right frontal lobe  and the grounding/reference electrode was placed in the cerebellum. Two EMG electrodes, gold plated, were lowered bilaterally into the neck muscle, directly caudal to the occipital bone. All EEG recording electrodes consisted of stainless-steel screws (Bilaney) with the following dimensions: head diameter 2.5 mm, shaft diameter: 1.57 mm, shaft length: 1.6 mm. All wires were connected to a head connector , which was secured over the skull using acrylic C and B Metabond (Parkell Inc.). Next, dental cement (Stoelting) was applied around the head connected to protect all the wires and the connector.The animals were anesthetized with a mixture of ketamine  to the sleep chambers  for 48-h after which, 24-h baseline (BL) EEG and EMG were recorded. Moreover, the pre-CSD homeostatic response following 4-h of SD was recorded for 21-h. After 15-d of CSD and SI test, EEG and EMG baseline (BL) recordings for 24-h were performed after 48-h of Hab in the sleep chambers. Additionally, the post-CSD homeostatic response following 4-h of SD was recorded for 21-h.After the postoperative recovery period, mice were transferred to a quasi-soundproof isolation sleep chamber  under the standard laboratory conditions . Mice were connected to a cable plugged to a rotating commutator  to allow free movement in all three dimensions during the chronic recording sessions (video monitored). Unipolar EEG and bipolar EMG signals were amplified 800× . The digitization was performed using a DAQ card . The data were sampled at 30 kHz. The videos were synchronized with the EEG recording via an output TTL signal (5V pulse) to trigger the start and the end of the video.The electrophysiological signals were filtered with a low-pass filter (cutoff frequency 7 kHz) and subsampled at 250 Hz. Next, the electrophysiological signals, the actimetry and the video were imported into a custom software program .The vigilance states Wake, NREM sleep and REM sleep were visually scored off-line using the EEG and EMG signals according to standard criteria and methods  with a 5The percent time, average bout duration and the number of bouts were extracted to quantitatively assess the vigilance states. Additionally, the spectral parameters of the EEG signal from right parietal electrode  were computed using custom software  using the mean spectrum analysis with rectangular windows with 20% overlap. Further analysis of the data was performed using scripts in Python.The 4-h SD was performed on the mice using the Viewpoint platform system at 7 AM (ZT 0). For this study, we used the basic SD paradigm consisting of sending random electric pulses in a randomized sequence to the magnet placed under the platform which pushes it up and wakes up the mice. The duration of a pulse is 1000 ms. The number of pulses per sequence is random between 3 and 5 pulses. The sequences of pulses were separated by a randomized time from 0.15 to 0.27 min. The mice were monitored using the video tracking system during the SD time. Moreover, EEG recording was performed during the 4-h SD paradigm.post hoc analyses on time series and average data, we used Tukey’s multiple comparisons test (p < 0.05). One sample t-tests were performed to measure changes of the 12-h average of % time, number of bouts and average bout duration across post- and pre-CSD.To compare the sleep behavior between the three phenotypes, we used mixed-effects model, instead of two-way repeated measures ANOVA, to account for the missing values . Time series of percent time, number of bouts, and average duration of bouts were created by quantifying and averaging across 2-h time intervals for a 24-h BL pre-CSD, 24-h BL post CSD and 20-h homeostatic sleep responses pre- and post-CSD. To compare between the different time series such as percent time, number of bouts, average duration of bouts of the three vigilance states , a mixed-model two-way ANOVA was performed with between-subjects factor “phenotype” (Susceptible vs. Resilient vs. Stress-naïve) and within-subjects factor “time” (12 × 2 h) for BL recordings. When a significant “phenotype” × “time” interaction was observed, a mixed-model two-way ANOVA was performed with between-subjects factor “phenotype” (Susceptible vs. Resilient vs. Stress-naïve) and within-subjects factor “time” (6 × 2 h) to investigate the light and the dark phases separately. For Post hoc analysis to assess the inter-phenotype difference was Turkey’s multiple comparisons test (p < 0.05) and for intra-phenotype difference across conditions or phases was SIDAK’s multiple comparisons test (p < 0.05).One-way ANOVA was performed to compare the bouts latencies and the average duration of inter-bout interval during the light and the dark phase pre-and post-CSD. Latency is defined as the time until the first bout of the different vigilance states. Then, a mixed-model two-way ANOVA was used to analyze across conditions to determine the between-subjects factor/interphenotype “phenotype” effect (Susceptible vs. Resilient vs. Stress-naïve) and within-subjects factor/intraphenotype effect such as “stress” (pre-CSD vs. post-CSD) or “phase” (Light vs. Dark). p < 0.05).To compare between the different power spectra, a mixed-effects model with between-subjects factor “phenotype” and within-subjects factor “frequency” (90 Hz × 0.5 Hz) was performed. Power spectra of NREM bouts were normalized to the pre-stress baseline average value of SWA (0.5–4.5 Hz) during the 12-h light phase. The low frequency range of normalized SWA (0.5–3 Hz) of NREM bouts, following 4-h SD, was quantified and averaged across 2-h time intervals. Two-way repeated measures ANOVA with “phenotype” as between-subjects factor and “time” (4-h × 2-h) as within-subjects factor was performed. Friedman’s Aligned Rank test was used to compare between the average cumulative duration of NREM and REM bouts of the homeostatic sleep response post SD (F-test) and Variance Inflation Factor (VIF) to detect collinearity between features were performed. Logistic regression model was built in Python using the Scikit-learn library. Cross-validation of the predictive model was performed by splitting the data into 75% training set and 25% test set.To build a predictive model for resilience and susceptibility using pre-stress sleep features, feature engineering using SelectKBest algorithm (p < 0.0001) for all vigilance states. There was an interaction between “phenotype” × “% time” of wake  the similar increases in the number of wake and NREM bouts, (ii) the decrease in the average duration of NREM bouts and (iii) the increased switching between NREM and wake bouts. This finding indicates that fragmented NREM sleep may be a predictor of vulnerability to future stress. Moreover, the increase in NREM fragmentation persisted post exposure to stress in susceptible mice. Interestingly, the mice resilient to stress displayed higher fragmentation of NREM, post exposure to stress, but only in the dark. We also assessed the latencies and average duration of interbout interval pre-and post-CSD stress . HoweverIt is worth mentioning that by combining the features of all vigilance states  of both susceptible and resilient mice, pre-CSD, into one category labeled stress-exposed mice, they became comparable to the features of the stress-naïve mice. This finding suggests that the sleep wake profile of stress-naïve mice likely consist of a combination of sleep wake profiles from both categories .p < 0.05; p < 0.05; p < 0.05 and p < 0.05, respectively; p < 0.05; p < 0.0001; Moreover, we were interested in assessing the difference in the homeostatic sleep response pre-and post-CSD in both the susceptible and resilient mice relative to stress-naïve mice. We investigated the homeostatic sleep response in mice after 4-h SD applied at the start of the light phase pre-CSD . There wF18,156 = 1.81, p < 0.05; F18,156 = 3.087, p < 0.0001; F2,18 = 6.098, p < 0.01; p < 0.05 and p < 0.05, respectively; p < 0.05 and p < 0.05, respectively; p < 0.05 for all comparisons; p < 0.05; p < 0.0001; p < 0.05; F1.732,29.45 = 4.672, p < 0.05; Next, we investigated the homeostatic sleep response in mice after 4-h SD applied at the start of the light post-CSD . There wF-test, by ranking them based on the ANOVA F-values  play a role in generating the down cortical state and the slow wave sleep (SWS) firing pattern  mice and stress-naïve mice. This suggests that the sleep and wake profile of stress-naïve group is made up of a combination of the sleep and wake profiles from both categories of stress-vulnerable and resilient mice. Therefore, some of the sleep features of the stress-naïve mice such as% Time, number of bouts and average bout duration might be a weighted “average” of the features from both susceptible and resilient mice. Further investigation is required to address such a possibility by applying non-supervised learning techniques on sleep-wake features from a greater number of stress-naïve mice than our current study, prior to stress, to investigate: (i) whether there is an inherent clustering into two phenotypes, (ii) the inherent ratio or representation of both vulnerable and resilient group features in the stress-naïve group, and (iii) whether factors such as differences in social hierarchy or early-life maternal stress lead to the generation of the sleep disturbances characterizing the stress-vulnerable phenotype.Pre-CSD, all three groups of mice spent the same amount of time in the different vigilance states during the light phase, while in the dark phase the stress-resilient mice slept less, relative to the stress-susceptible mice. Closer examination of the sleep architecture such as bout dynamics strength . Moreovestrength . The lea pattern . Thus, aPost-CSD, in the light phase, both resilient and susceptible-mice exhibited lower percentage of time in NREM sleep compared to stress-naïve mice. Moreover, the susceptible mice continued to maintain hyper-fragmented NREM sleep during both the light and dark phases, which is reminiscent of the high co-occurrence of insomnia (fragmented sleep) observed in patients with depression . Previoup = 0.056) and augmented REM sleep rebound, following SD, in the susceptible mice, post-CSD. However, the main contribution of our study is filling the gap in knowledge about the potential role of pre-existing sleep abnormalities in predicting vulnerability to stress. The increased fragmentation of NREM sleep that we observed in susceptible mice, prior to CSD exposure, is reminiscent of the role of insomnia and poor sleep continuity in increasing the risk for depression in humans. It is worth noting that there is a paradigm shift in research toward understanding the functional role of insomnia, instead of REM sleep aberrations, as a critical feature of depression .BR and DC designed the experiments and wrote the manuscript. BR and GJ collected the data. BR analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Periodontitis (PD) is a chronic inflammatory disease that can eventually lead to tooth loss. Genetic and environmental factors such as smoking are involved in the pathogenesis of PD. The development of PD is potentiated by various pathogens that induce an immune response leading to the production of cytokines, such as interleukin (IL)-17. The synthesis of IL-17 is influenced genetically. The polymorphisms in IL-17 gene may affect the synthesis of IL-17. The aim of this study was to examine the association between the IL-17F rs763780 and IL-17A rs2275913 polymorphisms and PD in non-smoking and smoking patients to check if these polymorphisms could be a risk factor for PD. Methods: The study enrolled 200 patients with PD (130 non-smokers and 70 smokers) and 160 control subjects (126 non-smokers and 34 smokers). Periodontitis was diagnosed on the basis of 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. All samples were genotyped using allelic discrimination assays with TaqMan® probes on a Real-Time PCR Detection System. Results: There were no statistically significant differences in the distribution of the IL-17F rs763780 and IL-17A rs2275913 genotypes and alleles between patients with PD and control subjects, between smoking patients with PD and smoking control subjects, and between non-smoking patients with PD and non-smoking control subjects. Conclusions: The results of this study suggest a lack of statistically significant associations between IL-17F rs763780 and IL-17A rs2275913 polymorphisms and PD in Polish population.  Periodontitis (PD) is characterized by inflammation and infiltration of inflammatory cells in periodontal tissue and continues to be a major public health problem . PreviouInterleukin (IL)-17 is a proinflammatory cytokine involved in the pathogenesis of PD and other inflammatory diseases . This cyThe interplay between environmental and genetic factors induces the development of PD . PreviouIL-17 genes functional polymorphisms (IL-17F rs763780 and IL-17A rs2275913) that alter mRNA and protein expression has been found [IL-17F rs763780 and IL-17A rs2275913 in PD patients to see if they could be a risk factor for the development of this disease in Polish population.Genes encoding IL-17A and IL-17F are located on chromosome 6 (6p12) [en found . Polymoren found ,29,30,31en found ,33,34,35en found . UnfortuThe patients from the West Pomeranian region of Poland, who came in years 2017–2019 to the Department of Periodontology with periodontitis had a medical interview and also a clinical and periodontal examination. Periodontitis was diagnosed on the basis of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions . This study included 360 Caucasian subjects: 200 patients with chronic periodontitis  and 160 healthy subjects without periodontal disease as a control group . Autoimmune diseases and diseases such as hepatitis, AIDS, diabetes or uncontrolled hypertension excluded patients from the study. Also the patients who used systemic antimicrobial agents, chronic anti-inflammatory medication, immunosuppressive medications and antibiotic within the past 6 months were excluded. All patients were otherwise healthy and had not been subjected to periodontal treatment before the study.The subjects were categorized into four subgroups: with and without periodontitis, according to the presence of smoking addiction . Smokers included people who had been continuously smoking at least 10 cigarettes a day for at least 5 years. The study was approved by the ethics committee at Pomeranian Medical University, Szczecin (number BN-001/93/08), Poland. All participants gave informed written consent to participate in the study. Clinical attachment loss (CAL) and probing pocket depth (PPD) were performed in each patient at six sites per tooth using a periodontal probe marked every 1 mm . Pressure of approximately 20 g was applied for probing. In addition, approximal plaque index (API) and modified sulcus bleeding index (mSBI) were calculated. Clinical measurements were performed by a periodontist specialist in each patient qualified for the study in the same conditions in a dental clinic—based on periodontal examination using the same instruments  and radiological examination (orthopantomogram). Periodontitis was detected as clinical attachment loss with a standardized periodontal probe with reference to the cemento-enamel junction. Patients were diagnosed with periodontitis if interdental CAL ≥ 2 was detectable at ≥2 non-adjacent teeth or buccal or oral CAL ≥ 3 mm and pockets > 3 mm were detectable at ≥2 teeth and the observed CAL couldn’t be attributed to non-periodontal causes. Radiographic bone loss had to be at least 15%. Extent and distribution of periodontitis was described as generalized when more than 30% of teeth were involved. ® probes  on a 7500Fast Real-Time PCR Detection System .All samples were genotyped in duplicate using allelic discrimination assays with TaqManp-value of < 0.05 was considered to indicate a statistically significant result. The study sample size was sufficient to detect with 80% probability the true effect size for comparison of allele frequencies between whole groups of PD patients and controls measured as odds ratio (OR) equal to 0.64 or 1.53 for rs2275913 and 0.18 or 2.51 for rs763780. The corresponding minimal detectable effect sizes  for subgroups of subjects stratified according to smoking status were: 0.59 or 1.65 for rs2275913 and 0.09 or 2.79 for rs763780 in non-smokers, and 0.37 or 2.33 for rs2275913 and 5.89 for rs763780 in smokers.The consistency of the genotype distribution with Hardy–Weinberg equilibrium (HWE) was assessed with Fisher’s exact test. Chi-square and Fisher’s exact tests were used to compare genotype and allele distributions between groups. A The clinical periodontal parameters in the studied groups are shown in IL-17F rs763780 and IL-17A rs2275913 genotypes among smoking and non-smoking patients with PD as well as smoking and non-smoking control subjects was in HWE and is shown in IL-17F rs763780 and IL-17A rs2275913 genotypes and alleles did not differ between patients with periodontitis and controls. The distribution of the IL-17F (rs763780) T and C alleles in total group of PD patients was 97.50% and 2.50% respectively and did not differ statistically significant from the control group (96.25% and 3.75%), OR—0.66, 95%CI—0.28–1.54, p = 0.39,  G and A alleles in total group of PD patients was 61.68% and 38.32% respectively and did not differ statistically significant from the control group (62.26% and 37.74%), OR—1.03, 95%CI—0.76–1.39, p = 0.88,  T and C alleles was 98.08% and 1.92%, respectively in non-smoking patients with PD, while in the non-smoking control group it was 96.03% and 3.97% respectively . In non-smoking patients with PD, the frequency of the IL-17A (rs2275913) G and A alleles was 57.42% and 42.58%, respectively, while in the non-smoking control group it was 60.80% and 39.20%, respectively , (The frequency of the 1–1.64), .IL-17F (rs763780) T and C alleles in smoking patients with PD was 96.43% and 3.57%, respectively, while in the smoking control group it was 97.06% and 2.94%, respectively . The frequency of IL-17A (rs2275913) G and A alleles in smoking PD patients was 69.57% and 30.43% respectively and did not differ significantly from the control group , (The frequency of the 9–1.71), .IL-17F rs763780 and IL-17A rs2275913 polymorphisms and PD to verify if these polymorphisms could be the risk factors for PD development. This analysis was performed separately in smoking and non-smoking patients and the control subjects. Tobacco smoking is one of the most important environmental risk factors for PD. Smoking may affect the host immune response to bacterial infections as well as the synthesis of proinflammatory mediators including cytokines [IL-17F (rs763780) C allele (OR—0.48) and increased frequency of IL-17A (rs2275913) A allele (OR—1.15). In the group of smoking patients, the results were opposite. We observed increased frequency of IL-17F (rs763780) C allele (OR—1.22) and decreased frequency of IL-17A (rs2275913) A allele (OR—0.92). These results suggest that the effect of IL-17 gene polymorphisms on PD risk may depend on smoking status. The pathogenesis of PD is very complex and involves interplay between genetic and environmental factors. It is likely that smoking may significantly alter the genetically determined synthesis of cytokines and the inflammatory response to bacterial infections in the periodontal tissues. In this study, we examined the relationship between the ytokines ,13,14,15IL-17 is a cytokine with a multi-directional action. It is involved in maintaining of barrier integrity in periodontal tissue and defence against pathogens. IL-17 induces the synthesis of antimicrobial mediators. On the other hand, these mediators enhance the inflammatory status in periodontal tissue and induce the development of periodontitis. Previous studies indicated the role of IL-17 in pathogenesis of PD. The studies have shown both increased and decreased expression of IL-17 in periodontal tissue in PD patients ,40,41,42IL-17 gene polymorphisms in patients with PD and the results are inconsistent. Saraiva et al. suggested that the IL-17A (rs2275913) A allele is associated with the lower frequency of PD in the Brazilian population [IL-17A (rs2275913) A allele is associated with an increased risk of chronic PD. In addition, the IL-17A (rs2275913) A allele was correlated with worse clinical parameters, higher myeloperoxidase activity, and increased expression of inflammatory mediators, compared with the other genotypes. Zacarias et al. [IL-17A rs2275913 and IL-17F rs763780 polymorphisms and PD in the Brazilian population. These authors showed that the IL-17A (rs2275913) AA genotype and the A allele were associated with increased susceptibility to chronic PD. In another study, Chaudhari et al. [IL-17A gene rs227591 polymorphism was associated with chronic and aggressive PD in the Indian population. The study by Jain et al. [IL-17F gene rs763780 polymorphism and PD in the Indian population. Borilova et al. [IL-17F rs763780 and IL-17A rs2275913 polymorphisms and PD in patients with type 1 diabetes. The IL-17A (rs2275913) A allele was associated with an increased production of IL-17 by mononuclear cells of patients with PD. In a meta-analysis, da Silva et al. assessed the association between IL-17F rs763780 and IL-17A rs2275913 polymorphisms and the risk of chronic and aggressive PD [Several studies have investigated pulation . Corrêa pulation  showed ts et al.  examinedi et al.  indicaten et al.  did not a et al.  examinedssive PD . This meIL-17 gene polymorphisms and the risk of PD. Our study is limited by the number of subjects. It is likely that these relationships could reach statistical significance in a larger cohort of patients. Our results suggest that the association between IL-17 gene polymorphisms and PD may depend on smoking status. The interaction between genetic and environmental factors leads to inflammation in periodontal tissue. Smoking may affect the neutrophil activity, cytokine synthesis and inflammatory status [The observed differences between above studies may be due to ethnic differences, different forms and stages of PD as well as smoking status of patients. We have not indicated statistically significant associations between y status ,13,14,15IL-17F rs763780 and IL-17A rs2275913 polymorphisms and PD in a Polish population. This association may depend on the smoking status of patients.The results of this study suggest a lack of statistically significant associations between


The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected persons of all ages, including the newborns. Few published case reports and case series have described the possibility of vertical transmission of COVID-19. In the present report, we describe a young primigravida at 33 weeks of gestation, who presented with a four-day history of low-grade fever, malaise, and breathing difficulty. She underwent testing of nasopharyngeal swab sample by real-time polymerase chain reaction (RT-PCR), which was positive for COVID-19. Cesarean section was done, and a preterm low birthweight baby was delivered. The baby required resuscitation at birth and was mechanically ventilated for a shorter duration. A tracheal aspirate that was taken at 12 hours of life tested positive for COVID-19. The course and outcome of the newborn are described here along with the possibility of vertical transmission. The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected persons of all ages, including the newborns . In thesA primigravida aged 30 years under quarantine complained of sore throat, low-grade fever, and malaise. She got tested and found positive for COVID-19. After two to three days, she developed breathing difficulty, decreased fetal movements, edema, and visual disturbance. Finally, she got referred to us for the subsequent management of her illness. She had already received two doses of TT (tetanus toxoid) injections and was on vitamin and mineral supplements  regularly. She was also taking thyroxine tablet (25 µg) once daily for hypothyroidism. She developed hypertension in the second month of pregnancy. Ultrasound done at 33 weeks of gestation revealed a single, live fetus (weight = 930 g), and the amniotic fluid index (AFI) was 9. She was conscious at admission with the following vital parameters: temperature 98.6˚F, pulse rate 112/min, respiratory rate 30/min, and blood pressure 190/110 mmHg. General examination showed pallor, edema, and icterus. There was 50 ml of bloody urine in the urinary bag. She was finally diagnosed to have HELLP syndrome with hypothyroidism and moderate COVID-19 pneumonia  was started without delayed cord clamping and skin-to-skin contact. A chest X-ray done was normal  was delivered. The newborn did not cry immediately , and required resuscitation. Mechanical ventilation . There was no further recurrence of seizure. As the condition of the newborn improved, he was extubated to continuous positive airway pressure (CPAP) after 24 hours of mechanical ventilation. RT-PCR of the tracheal aspirate sample that was taken after 12 hours found positive for COVID-19. However, he showed consistent improvement, was weaned off oxygen, and discharged in a healthy condition.While reviewing the literature, we could find a few published case reports and case series on neonatal SARS-CoV-2 (COVID-19) infection. The authors in one review reported 179 cases of newborns tested positive at birth, whose mothers were infected in the third trimester of pregnancy . The timTo date, there is a lack of clarity regarding the criteria of congenital COVID-19 infection. The authors of one study described that three newborns born to mothers with COVID-19 infection had positive antibodies (IgM and IgG) at birth ,8. WhethThe present report has some limitations. Due to lack of logistics, the presence of the COVID-19 virus was not confirmed in body fluids  or tissue specimens  that might have further clarified the mode of transmission. In addition, COVID-19 antibody testing also could not be performed in the newborn.Our report shows that COVID-19 infection may possibly get transmitted vertically to the fetus. More research is needed to better elucidate the mechanisms involved in this process.


In addition, the effective connectivity from middle occipital gyrus  to insula is also increased in patients as compared with healthy controls. Aberrant insula to executive network influence is pronounced in patients with more severe negative symptom burden. These findings suggest that control signals from rAI are abnormally elevated and directed towards both task-positive and task-negative brain regions, when task-related demands arise in schizophrenia. This aberrant, undiscriminating surge in salience signalling may disrupt contextually appropriate allocation of resources in the neuronal workspace in patients with schizophrenia.Triple network dysfunction theory of schizophrenia postulates that the interaction between the default-mode and the fronto-parietal executive network is disrupted by aberrant salience signals from the right anterior insula (rAI). To date, it is not clear how the proposed resting-state disruption translates to task-processing inefficiency in subjects with schizophrenia. Using a contiguous resting and 2-back task performance fMRI paradigm, we quantified the change in effective connectivity that accompanies rest-to-task state transition in 29 clinically stable patients with schizophrenia and 31 matched healthy controls. We found an aberrant task-evoked increase in the influence of the rAI to both executive (Cohen’s  In thisImpaired rAI modulation of CEN and DMN observed using resting-state fMRI relates to impaired cognitive task performance across various psychotic disorders . To dateCognitive deficits involving working memory are tightly linked to the domain of negative symptoms characterised by psychomotor poverty and persistent functional disability in schizophrenia. While the mechanism of negative symptoms continue to be elusive, 22.1The sample consisted of 32 patients satisfying DSM-IV criteria for schizophrenia (SZ) or schizoaffective disorder and 32 matched healthy controls (HC) reported in our prior studies . Patient2.22.2.1As described in our prior reports  functionA magnetization-prepared rapid acquisition gradient echo image with 1 mm isotropic resolution, 256 × 256 × 160 matrix, Repetition Time (TR)/Echo Time (TE) 8.1/3.7 ms, shot interval 3 s, flip angle 8°, SENSE factor 2 was also acquired for each participant for volume registration. Weighted summation of the dual-echo images produced a single set of low-artefact functional images .2.2.2We used a visual n-back task with a button press response in two sessions of fMRI recording. Seven task blocks each of 110 s duration were presented in each session. Each task block consisted of 0-back, 1-back, and 2-back conditions of 30 s duration. Each condition presented in a random sequence, with 10 s interval between the conditions. On-screen instructions preceded every condition indicating the type of response required . Each condition included four target and 11 non-target stimuli with a 2s inter-stimulus interval. To ensure adequate task comprehension and performance, all participants performed a practice version of the task outside the scanner prior to scanning. All scanned participants successfully identified in excess of 80% of targets in the practice task. Task sessions immediately followed the resting sessions during the acquisitions See .2.2.3www.fil.ion.ucl.ac.uk/spm) and DPABI: a toolbox for Data Processing & Analysis for Brain Imaging (rfmri.org/dpabi)  . For theg/dpabi) .For task fMRI data, we used an additional task regressor per condition convolved with a hemodynamic response function (with default parameters in SPM8) to control for the false connectivity owing to the common associations with the task design .Removal of global signal has been shown to reduce physiological noise, including those arising from motion both at rest  and duri2.32.3.1We defined a seed-region as a 6 mm-sphere  for the right anterior insula [rAI] following our previous study of the SN in SCZ using resting-state fMRI . We condhttps://github.com/qluo2018/Resting2TaskShifting.For the resting scans, we estimated the path coefficient [PC] from the seed to the whole brain by GCM . For theWe estimated the change of the path coefficient [CPC] across all voxels [CPC maps] contrasting resting from task-active state (seed-to-whole brain and vice versa).2.3.2t-test after adjustment for the covariates age, gender, and head movement (meanFD). Clusters with significantly different CPC were identified after the updated version  on the findings. To determine whether the identified increase in CPC meant a larger positive effect or a change from negative effect to positive effect, we carried out additional one-sample t-test for the significant clusters considering the same set of covariates.The estimated CPC maps were compared between patients and controls by two-sample  version  of Alpha2.3.3p < 0.025 (two-tailed). For other exploratory correlations, we used an uncorrected threshold (p < 0.05).We averaged the CPCs among all voxels within each significant cluster, and tested the behavioural and symptom associations of the CPC at cluster level. Spearman correlation was used for behavior score (Hit rate of task performance) and three symptom scores , while Pearson’s test was used for the Social and Occupational Functioning Assessment Scale SOFAS;  and Sign33.1T54 = 3.93, p = 2.4 × 10−4 at peak voxel), left precuneus (PCUN)  and right middle occipital gyrus (MOG) , exhibited significant differences in CPC between SZ and HC groups  , as the mean statistics had less variation than the peak  in patients with SZ compared to HC  influence on rAI during task compared to rest, while patients showed a non-significant increase  in influence from left MOG to rAI.With rest-to-task contrast, the path coefficients from the visual cortex (left MOG) to rAI increased , left SFGdor , right ANG , right MTG  and right SMA ] CPC compared to HC groups  with task performance (the hit rate of 2-back task), while the CPC from rAI to left PCUN was negatively correlated  with the task performance. These associations were significant  even after adjusting for covariates . However, both associations were absent in patients . The reduced CPC from right SMA to rAI was associated with lower SOFAS . There were no correlations between CPC values of any of the clusters and current or cumulative life time exposure to antipsychotics .In patients with schizophrenia C and D, 3.4The group-difference in CPC remained significant even after controlling for the brain activation of the seed region and the identified cluster (see Supplement Table S8). We further demonstrated the specificity of our results to one-TR lag GCA of rAI by contrasting this with GCA of a posterior insula seed, as well as computing the change coefficient for functional connectivity of the rAI seed. We also confirmed that the change in effective connectivity of rAI to the MFG and PCUN nodes, as well as SMA to rAI were disrupted but less pronounced with the 1-back compared to the 2-back load effect (see Supplement Tables S9–S11).4d = 1.35) and precuneus (d = 1.22), alongside a task-related decrease in inflow into rAI from distributed brain regions in schizophrenia. In healthy controls, lower n-back performance was related to a task-related reduction in rAI-to-MFG but an increase in rAI-to-PCUN effective connectivity. However, despite significantly lower performance, patients had a non-selective increase in rAI’s influence on both MFG and PCUN. These observations confirm our primary hypothesis that control signals from rAI are abnormally elevated and directed towards both task-positive and task-negative brain regions, when task-related demands arise in schizophrenia. This aberrant surge in proximal salience  to DMN and CEN may indicate a system-level failure of resource allocation when cognitive demands arise  cortex has a specific task-related divergence in effective connectivity to and from the rAI in schizophrenia. This task-related increase in MOG-to-rAI influence occurs in sharp contrast to the generalised reduction in influence from other brain regions to rAI in schizophrenia. Notably, this increase in MOG-to-rAI influence occurs in patients irrespective of their symptomatic and functional status, indicating a breakdown of a crucial bottom-up pathway that feeds sensory information to the salience network . MechaniWe noted higher task-related outflow signal from rAI-to-MFG (and lower rAI-to-PCUN) was associated with higher hit rate in healthy controls, indicating that differential signalling from rAI-to-DMN/CEN nodes may serve to dissociate the two competing information processing systems and thus increase the accuracy of stimulus–response association. This observation is in line with several studies that note that the functional divergence of DMN and CEN aids in task performance . On the We noted that the rAI-to-MFG outflow, but not the rAI-to-precuneus outflow, was pronounced in patients with a high degree of psychomotor poverty (negative symptoms), consistent with a prior report  using inOur study has several strengths. Notably, we used an approach of effective connectivity that aids us to parse directionality of network connectivity. Further, by employing a distinct 10-minutes long resting session followed by the task paradigm, we avoided task-related spill-over effects on the resting data. There were several limitations as well in the current study. We studied a sample of medicated patients, which may limit our results since dopamine-blocking agents are known to affect connectivity patterns . NeverthWe conclude that the aberrant distribution of salience signals generated by the SN disrupts the discriminatory neural processes required for contextually relevant responses, providing a parsimonious explanation for the neurocognitive deficits that lie at the core of schizophrenia. Approaches that directly modulate the effective connectivity of anterior insula , could pMedical Research Council, UK (grant number G0601442). LP is supported by the Tanna Schulich Endowment Chair, the Academic Medical Organization of South-Western Ontario (Opportunities Fund) and the 10.13039/100009007Canadian Institutes of Health Research Foundation Grant (grant number 375104). QL was supported by the 10.13039/501100001809National Natural Science Foundation of China , Shanghai Municipal Science and Technology Major Project (grant number 2018SHZDZX01), and Zhangjiang Lab. HG was supported by the 10.13039/501100001809National Natural Science Foundation of China (grant numbers 11872276 and 11572225). PFL receives research funding from NIHR Nottingham Biomedical Research Centre.This work was supported by the DisclosuresDr Palaniyappan reports speaker fees from Janssen and Otsuka Canada, Canadian Psychiatric Association and SPMM Course (UK); investigator-initiated educational grants from Janssen Canada, Otsuka Canada and Sunovion not related to the submitted work. The other authors reported no biomedical financial interests or potential conflicts of interest.L.P. and P.L. conceptualized the study. Q.L and L.P. designed the methodology. Q.L., B.P., H.G., and L.P performed most of the analyses. M.S., S.F., P.L. and L.P. acquired and processed neuroimaging, behavioural and clinical data. Q.L., B.P., and L.P. wrote the manuscript. H.G., M.S., S.F., and P.L made critical revision of the manuscript. P.L., L.P. and Q.L. acquired the funding. All authors reviewed the manuscript and discussed the work.


Background. Atrial fibrillation (AF) increases the risk for stroke but also for non-stroke major adverse cardiovascular events (MACE). The 2MACE score was recently proposed to predict these events. Since the interest of microRNAs (miRNAs) in cardiovascular diseases is increasing, we aimed to investigate whether miRNA levels may improve the predictive performance of the 2MACE score. Methods. We included consecutive AF patients stable on vitamin K antagonist therapy. Blood samples were drawn at baseline and plasma expression of miRNAs was assessed. During a median of 7.6 (interquartile range (IQR) 5.4–8.0) years, the occurrence of any MACE  was recorded. Results. We conducted a miRNA expression analysis in plasma from 19 patients with and without cardiovascular events. The miRNAs selected  were later measured in 166 patients  years) and all were associated with a higher risk of MACE. The addition of miR-107 and miR-146a-5p to the 2MACE score significantly increased the predictive performance , and the model with three miRNAs also improved the predictive performance compared to the original score . 2MACE models with the addition of miRNAs presented higher net benefit and potential clinical usefulness. Conclusions. Higher miR-22-3p andmiR-107 and lower miR-146a-5p levels were associated with a higher risk of MACE. The addition of these miRNAs to the 2MACE score significantly increased the predictive performance for MACE, which may aid to some extent in the decision-making process about risk stratification in AF. Atrial fibrillation (AF) is the most frequent cardiac arrhythmia and it implies a high morbidity and mortality ,2. ThereOn the other hand, the study of biomarkers as prognostic or risk stratification markers has gained attention. In this context, microRNAs (miRNAs) are post-transcriptional regulators that have been widely recognized as active effectors of the cardiovascular system . The recIn the present study, we aimed to determine a profile of circulating miRNAs with the ability to predict MACE in AF, and to assess whether plasma miRNA levels can improve the predictive performance of the 2MACE score in a “real-world” cohort of AF patients under vitamin K antagonist (VKA) therapy.We included a prospective cohort of permanent/paroxysmal AF patients from our outpatient anticoagulation clinic in a tertiary hospital . All patients were on VKA therapy with stable international normalized ratios (INR 2.0–3.0) during at least the previous 6 months. This 6-month period of good anticoagulation control with VKA before inclusion was required to have a baseline homogeneity that would allow us to avoid the potential bias of poor anticoagulation control on clinical outcomes. Patients with prosthetic heart valves, rheumatic AF, or patients with acute coronary syndrome, stroke (ischemic or embolic), unstable chest pain, hemodynamic instability, hospital admission, or surgical intervention in the preceding 6 months were excluded.2DS2-VASc) and bleeding risk (HAS-BLED) were estimated. The time in the therapeutic range at 6 months after entry was calculated using the Rosendaal method.The recruitment period was from May 2007 to December 2007. At inclusion, a complete medical history was recorded, and stroke risk  and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its subsequent amendments.The baseline 2MACE score was retrospectively calculated in all patients as described by Pastori et al. . This scg and 4 °C for 10 min, and the supernatants were stored in aliquots at −80 °C until further use. miRNAs from plasma were purified using the Nucleo Spin miRNA Plasma Kit (Macherey–Nagel) in accordance with the manufacturer’s protocol. Plasma miRNAs were screened using the plasma/serum focus microRNA PCR Panel V4 (Exiqon) composed of 179 miRNAs as has been previously described [p-value < 0.0001) and r = 0.8902 , respectively. Plasma expression of miRNAs was calculated using the 2−ΔCt method.Blood samples were atraumatically drawn at study entry and without stasis into syringes preloaded with trisodium citrate (0.011 mol/L), i.e., all patients were stable under VKA therapy for at least 6 months when the blood sample was collected. Samples were centrifuged at 2200× escribed . Data weThe primary endpoint was the occurrence of any MACE , during the follow-up period. We excluded from MACE all embolic events; i.e., stroke, transient ischemic attack, and peripheral or systemic embolism.The median follow-up was 7.6 (interquartile range (IQR) 5.4–8.0) years. Follow-up was performed through routine visits to the anticoagulation clinic, the hospital electronic medical records, or, when unavailable, by telephone interview. No specific visits were performed regarding the study. Of note, no patient was lost to follow-up. The investigators identified, confirmed, and recorded all adverse events and outcomes.U test or the Student t-test, as appropriate.Continuous variables were presented as mean±SD or median (interquartile range (IQR)), according to the Kolmogorov–Smirnov test. Categorical variables were presented as absolute frequencies and percentages. The Pearson χ2 test was used to compare frequencies and comparisons of miRNA levels were analyzed using the Mann–Whitney Cox regression analyses were performed to investigate the association of each miRNA with the risk of MACE.Receiver operating characteristic curves were used to evaluate the predictive ability (expressed as c-indexes) of the original 2MACE score and the miRNA-modified ones. The cut-off point of each miRNA with the best combination of specificity and sensitivity was assessed by the Youden index. Comparisons of receiver operating characteristic curves were performed as described by DeLong et al. .Discrimination and reclassification performances were evaluated by calculating the integrated discrimination improvement (IDI) and the net reclassification improvement (NRI), according to the methods of Pencina et al. .x axis) and the net benefit of using the model to stratify patients at risk (y axis) relative to assuming that no patient will have an adverse event. Here, those models that are the farthest away from the slanted dashed line  and the horizontal line  at a particular threshold probability demonstrate the higher net clinical benefit.The clinical usefulness and the net benefit of the original score in comparison with the miRNA-modified scores was estimated by using the decision curve analysis (DCA), as was proposed by Vickers et al. . The DCAp-value < 0.05 was accepted as statistically significant. Statistical analyses were performed using SPSS 21.0 , MedCalc v. 16.4.3 , STATA v. 12.0 , and survIDINRI package for R v. 3.3.1 for Windows.A To test if miRNA levels were associated with the development of cardiovascular events, we firstly conducted a miRNA expression pattern analysis in plasma from 9 patients with and 10 patients without cardiovascular events. Since ischemic stroke is the classic main efficacy outcome in AF, we initially selected this endpoint to homogenize the samples. As shown in 2DS2-VASc and HAS-BLED were 4 (IQR 3–5) and 2 (IQR 2–3), respectively, whereas the median 2MACE score was 2.5 (IQR 1–4). A summary of baseline clinical characteristics is shown in The three miRNAs selected were measured in 166 patients  years). The median CHAWe first aimed to find an association between the levels of these three miRNAs and MACE in the cohort of 166 patients. During a median of 7.6 (IQR 5.4–8.0) years, 49  patients suffered a MACE. As shown in When the predictive ability for MACE was tested, receiver operating characteristic (ROC)curve analyses demonstrated that miR-22-3p and miR-107 presented poor c-indexes  whereas miR-146a-5p exhibited a moderate c-index . In order to test if any of these miRNAs could enhance the predictive ability of the 2MACE score, we identified cut-off points with the best combination of sensitivity and specificity. The best cut-offs were 0.191, 0.137, and 1.979 for miR-22-3p, miR-107, and miR-146a-5p, respectively. Therefore, patients with miR-22-3p or miR-107 levels over the cut-off points and patients with miR-146a-5p levels under the cut-off point were categorized as being at “high risk” of MACE.p = 0.004). Although miR-22-3p alone showed a poor c-index, we also tested a model with this miRNA in addition to miR-107 and miR-146a-5p into the 2MACE score. Similarly, this model also improved the predictive performance of the original 2MACE . In both models of miRNAs, the sensitivity was also enhanced, as is shown by the results of IDI  .In this study including steadily anticoagulated AF patients taking VKAs, we observed that plasma levels of miR-22-3p, miR-107, and miR-146a-5p were associated with a higher risk of MACE. Importantly, the predictive ability of the 2MACE score for this event was enhanced with the addition of miRNAs.During the last years, the interest of miRNAs in AF is increasing ,22,23. D2DS2-VASc score. This suggests that this miRNA is more expressed in patients with high thromboembolic risk, which could provide some information about the pathophysiological conditions of AF patients [For example, miR-22-3p showed to play a role in several cardiovascular diseases . Thus, apatients . Previoupatients . This ispatients ,34. It iIn another recent study, miR-107 was investigated in AF patients undergoing catheter ablation. Although this miRNA was downregulated in sera from patients with AF recurrence compared to patients without AF recurrence, differences in expression were not significant . HoweverThere is, however, more prognosis information regarding miR-146a-5p. A preliminary analysis of patients from this cohort at ~3 years of follow-up already showed an association of miR-146a-5p with adverse cardiovascular events . In that study, lower levels of miR-146a-5p due to the presence of a functional single-nucleotide polymorphism rs2431697 were related to poor prognosis . These dNevertheless, the clinical usefulness of biomarkers when they are evaluated alone may be only limited. In fact, a common criticism of biomarkers is their high variability and lack of specificity in different clinical settings ,39,40. HThere are some limitations that we aim to acknowledge. First, this study was performed in a single center, and it was composed of a Caucasian-based population. Second, all patients were hemodynamically stable and had good anticoagulation control during the previous 6 months after entry, in an attempt to homogenize the sample and avoid the potential impact of acute conditions or poor anticoagulation control in the development of adverse events. Therefore, our results might not be extrapolated to different clinically contexts. In addition, our study is limited only to AF patients treated with VKAs, since all patients were under this therapy at inclusion. Although VKAs are still the most commonly prescribed oral anticoagulants in Spain, the use of direct-acting oral anticoagulants (DOACs) is increasing worldwide. Thus, our results require further validation in AF patients under VKAs and DOACs in order to broaden their clinical usefulness. Finally, we only measured miRNAs at inclusion, and no other determinations have been performed throughout the follow-up. Comorbidities change over time and how miRNAs could evolve during the follow-up period has not been determined. For the above reason, further studies are warranted to confirm our results.In this study including AF patients taking VKAs, we found that higher miR-22-3p and miR-107 and lower miR-146a-5p plasma levels were associated with higher risk of MACE at follow-up. The addition of these miRNAs to the 2MACE score significantly increased the predictive performance for MACE and enhanced its clinical usefulness. A combination of miRNAs and clinical risk factors could aid to some extent in the decision-making process about risk stratification in patients with AF.


Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Activated L-PRP has an average of 3442 ± 2061 pg/mL of PDGF-BB.There are few publications on the subject of canine platelet concentrates and further studies are required to characterize these for clinical applications. The aim of this study determined platelet, erythrocyte, and leukocyte counts in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system . Whole blood (WB) from 30 healthy dogs was used. In 10 L-PRP bovine thrombin activated samples platelet-derived growth factor isoform BB (PDGF-BB) concentration was also measured. The CPUNT 20 produced clinically useful quantities of sterile canine L-PRP containing a high concentration of platelets , with a 4.4 fold increase in platelet count, lower concentration of erythrocytes  and similar concentration of leukocytes  compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Conclusion: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.Background: To characterize the cellular composition  and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. Methods: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system  according to the manufacturer’s instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. Results: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL,  Platelet-rich plasma (PRP) is a product derived from whole blood, characterized by platelet (PLT) concentrations higher than baseline in a small volume of plasma . Growth In human medicine various authors have tried to define simple parameters for assessing the quality of platelet concentrates, a difficult target to achieve. Marx  has repoThe collection and processing of PRP should preserve the platelets in a quiescent state so that activation and subsequent growth factor release will be delayed until the moment of clinical use . An impop-selectin externalization when PRP is activated [Despite the popularity and advantages of PRP, there are conflicting reports in the literature regarding its clinical efficacy ,47,48,49ctivated ,63,64,65ctivated  and moreThe aim of this study was to characterize the cellular composition  and determine the platelet-derived growth factor-BB (PDGF-BB) concentration after activation in the final product from a commercial semi-automated platelet-rich plasma closed system using canine blood.After approval by the Ethics Committee of the University of Milan (protocol number 13-01-15) and with owner-informed consent, citrated blood samples were collected from the cephalic vein of 30 healthy un-sedated adult canine blood donors (PLT count within the reference range), 17 males and 13 females, weighing between 20 and 45 kg and between 2 and 8 years of age (mean ± standard deviation: 4.2 ± 1.9 years). A variety of breeds were represented  and all dogs were admitted to the Veterinary Transfusion Research Laboratory (REVLab) of the Department of Veterinary Medicine for routine blood donor check-ups. All dogs were fasted for 12 h before blood sample collection.A closed semi-automatic platelet-rich plasma (PRP) collection and producing system for veterinary use  was used, according to the manufacturer’s instructions. Briefly, the system consists of a sterile, single use, disposable collection kit for blood sampling, a dedicated centrifuge and an automatic instrument for the separation of the PRP .The collection kit comprises a butterfly needle (19G) connected to a 20 mL syringe for blood aspiration, with a port for anticoagulant addition and an antibacterial filter to preserve kit sterility, and a 10 mL bag for the storage of the PRP . All thrTo prevent blood clotting, before blood sampling the plastic clip of the anticoagulant port was opened, 3 mL of 3.8% sodium citrate (not provided by the manufacturer) was inserted with the provided 10 mL sterile luer-lock syringe through the antibacterial filter and transferred to the aspiration syringe. The connection between the 2 tubes was then temporarily clipped.The plastic clip of the blood aspiration port was opened and whole blood (WB) was collected from the cephalic vein of each subject up to the 20 mL mark on the aspiration syringe. The collection route was then clipped, the anticoagulant port was opened and another 1 mL of 3.8% sodium citrate was inserted.g for 15 min, using the dedicated centrifuge equipped with special adapters. Immediately before centrifugation, the aspiration syringe was temporarily disconnected from the storage bag to withdraw 500 microliters of citrate WB for subsequent laboratory analysis. This step was performed only for the purposes of the study.After sample collection, the part of the kit with the butterfly and the anticoagulant port and a portion of the aspiration syringe piston were removed retaining only the part of the kit dedicated to the production of the PRP, i.e., the aspiration syringe connected to the 10 mL storage bag . These wAt the end of the centrifugation the syringe, in which erythrocytes, buffy coat and supernatant plasma layers are clearly visible, was gently removed from the adapter and the separating plastic clip between syringe and storage bag was opened. At this point, the kit was positioned in the automatic separation instrument. The movement of a plunger positioned under the syringe piston of the syringe under control of an optical reader isolated the supernatant plasma, the buffy coat and the surface of the erythrocytes layer into the storage bag . The aspg for 5 min, using the appropriate slots of the same dedicated centrifuge. At the end of this second centrifugation, a pellet suspended in the platelet poor plasma (PPP) was formed inside the storage bag  thus obtained  was collFor each test subject (30/30), platelet count (PLT/μL) leucocyte count (WBC/μL) and erythrocyte count (RBC/μL), were calculated on WB and L-PRP by an automatic analyzer using optical and volumetric impedance measurements , while neutrophils (cells/μL), monocytes (cells/μL) and lymphocytes (cells/μL) counts were calculated with the same instrument and checked with a manual differential count of blood smears in only 19/30 subjects, for technical reasons. In the same 19 subjects the smears were also used for assessment of possible platelet clumping. All sample were stored at room temperature on a laboratory blood rocker for a minimum of 5 min before counts were performed.This study was performed using a closed system designed to guarantee the sterility of the finished product. However, having temporally disconnected the aspiration syringe from the storage bag to withdraw 500 microliters of citrated WB for subsequent laboratory analysis (step not required by the method) and still wanting to test the validity of the method for further possible clinical uses, we performed bacteriological tests on seven, randomly chosen, L-PRP samples. One hundred microliters of L-PRP was plated by streaking onto blood agar plates  both for aerobic and anaerobic bacteria and then incubated at 35 ± 2 °C for at least 48 h .The PDGF-BB levels in 10 L-PRP samples were assessed after L-PRP activation of samples with. bovine thrombin. To each 1 mL of L-PRP, 50 μL of a bovine thrombin solution  containing 500 IU/mL was added. After activation, the samples were kept at 37 °C in an incubator. The supernatant (after spontaneous clot retraction) of each activated L-PRP was collected at 3 h after activation and the concentration of PDGF-BB (pg/mL) was determined using a human ELISA kit  previously validated in dog  and usedt-test depending on data distribution. The statistical differences between mean male and female PLT values were compared using Mann-Whitney test. The increase in platelet concentration in L-PRP over whole blood baseline values was calculated using the following equation: platelet count L-PRP/platelet count WB. Spearman’s coefficient of rank correlation (rho) was used to evaluate the relationship between cellular counts  in WB and in L-PRP. Correlation between cellular  and PDGF-BB concentrations in L-PRP were determined using Pearson test. For all tests significance was set at p < 0.05. Statistical analyses were performed using commercial software .The normal distribution of parametric data was calculated using the D’Agostino-Pearson test and only RBC and lymphocytes values were found to be normally distributed. Results are presented as mean ± standard deviation. The statistical differences between mean values of PLT, WBC, neutrophils, lymphocytes, monocytes and RBC on WB and L-PRP were compared using Wilcoxon rank sum test or paired p = 0.44). The mean PDGF-BB concentration in L-PRP samples after thrombin activation was 3442 ± 2061 pg/mL. Pearson correlation coefficient between cellular  and PDGF concentrations in L-PRP were 0.62 (p = 0.05), 0.60 (p = 0.06), 0.43 (p = 0.22), 0.50 (p = 0.14), 0.88 (p = 0.0008) and 0.23 (p = 0.52), respectively. All samples of L-PRP tested for bacteriological analysis were sterile.The mean volume of L-PRP obtained was 2.3 ± 0.7 mL. The average PLT, neutrophils, lymphocytes, monocytes and RBC values comparing WB and L-PRP were significantly different . There wIn this study canine L-PRP was prepared using a commercial semi-automated closed system. All the steps of the production method were easy to perform and the volume of platelet concentrate produced was more than sufficient (on average > 2 mL per subject were obtained) for therapeutic use: PRP has been used in quantities ranging from 0.6 to 3 mL in a variety of canine pathologies ,72,73,74The fact that the system is closed means the final product is free from bacterial contamination, as confirmed by our microbiological evaluation This cannot be guaranteed in other double centrifugation methods with the exception of a laminar flow chamber under sterile conditions and with trained personnel. This is particularly important when canine platelet concentrates will be used therapeutically ,60. The Interestingly, it is also possible to withdraw an aliquot of the L-PRP obtained from the collection bag in a sterile manner, so as to be able to keep the remainder for dermatological or oculistic periodic therapeutic treatments in the same subject. Shelf life of the remaining product may be prolonged by freezing, as carried is out in human medicine ,76, thoup < 0.0001). Traditionally, a three- to five-fold increase in platelets has been considered an appropriate concentration for medical applications [3 platelets/μL to be in the therapeutically effective range and this was reached by the seven sampled in this study that failed to concentrate 4-fold. This proposed minimum platelet number for PRP has been extrapolated from the human literature and key species differences in average platelet counts and platelet physiology preclude an accurate evaluation of literature data [The system utilized demonstrated a consistent ability to enrich platelets in autologous plasma (about fourfold) producing a significantly higher platelet count in L-PRP than in WB . Reducing RBC concentration is thought to be important when developing the ideal PRP product [In our study, the L-PRP system utilized significantly decreased the RBC concentration compared with WB ( product . A recen product .p = 0.03). The average WBC in our L-PRP did not reach the limit of twice baseline value defined by some authors [The CPUNT 20 separation instrument has two modes of use: the production mode of the PRP with the inclusion of leukocytes, which allows L-PRP (leukocyte- and platelet-rich plasma) collection and the production mode without the inclusion of leukocytes, which results in production of P-PRP (pure platelet-rich plasma), according to Dohan Ehrenfest’s classification. In this study we used the PRP production modality with the inclusion of leukocytes, obtaining an L-PRP with an average WBC not statistically different from that in WB, with a moderate positive correlation between WBC concentration in WB and L -PRP . The potentially deleterious effects of leukocytes are largely attributed to the presence of neutrophils [In our study, in L-PRP we achieved a decrease in neutrophils and an increase in lymphocytes and monocytes statistically significant compared to WB, with a positive correlation between lymphocytes concentration in WB and L-PRP . The relationship between platelet and PDGF concentrations is far from clear. Some authors report correlation between platelet and PDGF concentrations [p = 0.06 and 0.88 with p = 0.0008, respectively) in our results seems to corroborate the theory that the quantity of white blood cells in the PRP also influences the concentration of growth factors [This study shows a positive and significative correlation between PDGF-BB concentration and PLT concentration in L-PRP (Pearson index 0.62 with trations ,60 and otrations ,37,42,64trations  suggest trations ,102. The factors ,63,91.p-selectin dosage [A limitation of our study was that we did not measure PDGF-BB concentrations in whole blood. We could speculate that the concentration of PDGF-BB might increase in L- PRP, after activation, in our samples by comparing our values with those found in WB in the literature , but we n dosage ,64. Amonn dosage  and thern dosage .Another limitation of our study was that we did not evaluate some key inflammatory markers, such as interleukin—1β or TNF-α, and their correlation with WBC and PLT content in L-PRP. The concentration of these catabolic cytokines, that seems differ in various PRPs, may be clinically relevant. A final limitation was that we didn’t evaluate the production mode without the inclusion of leukocytes of the CPUNT 20 separation instrument, which results in production of P-PRP, according to the manufacturer’s instructions; it would have been interesting to compare the characteristics of the two options provided by the kit with canine samples.The closed semi-automated system utilized in this study produced a fourfold mean increase in platelets, but with a large standard deviation and reduced the relative percentage recovery of erythrocytes, with a similar concentration of leukocytes but with a lower concentration of neutrophils as in baseline WB. The autologous L-PRP obtained had PDGF-BB concentration comparable to previous data in the literature after activation with bovine thrombin, was sterile and was produced in a sufficient volume to allow clinical use in various fields of canine medicine. Further studies are recommended to evaluate inflammatory markers and clinical application of this L-PRP product in dogs.